Our goal is to understand the regulation of intracellular pH (pHi) in nere cells. The project is divided into two related parts. The first is a continuation of current work on the ionic mechanism pHi regulation in the squid axon. The axon's pHi is regulated by a Na-dependent Cl-HCO3 exchanger, which responds to intracellular acid loads (i.e., decreases in pHi) by increasing the rate at which it extrudes acid equivalents from the cell. The aims are to examine (la) the exchanger's thermodynamics while operating in reverse, (lb) the exchanger's dependence (operating in the forward direction) on (ATP)i, (lc) its dependence on pHi, its dependence on (ld) (C1-)i, and (le) the effect of increased (Mg++)i. (la) and (lb) will provide crucial information on the role of ATP. (lb) through (ld) will provide an important test of the NaCO3 ion-par model, and provide the first detailed information on the dependence of the exchanger on intracellular substrates/cofactors. (lc) and (le) may provide insight into how the exchanger is regulated by pHi. The experiments will be carried out on internally dialyzed squid axons, the pHi of which will be measured with microelectrodes. A newly developed intracellular-pH-clamp technique will be used to directly measure rates at which acid equivalents are extruded from the cell. The second part of the project is the first study of pHi regulation in mammalian neurons. We will isolate identified neurons (CA1 cells) from a specified region of the cerebral cortex (hippocampus) of adult guinea pigs and, on the same day, measure their pHi with a pH-sensitive fluorescent dye. The aims are to (2a) examine the cells for the presence of non-HCO3 acid-base transport systems; (2b) examine the cells for the presence of HCO3 acid-base transport systems; (2c) determine the role of each of these acid-base transport systems by examining the cells' acute and chronic responses to six fundamental extracellular acid-base disturbances; (2d) examine the effect on pHi of other experimental maneuvers; and (2e) extend the aforementioned studies to other cells of the central nervous system, both freshly isolated cells and those in long-term primary culture. This work is significant because the acid-base physiology of neurons is likely to play a key role in the growth and development of the nervous system, the function and regulation of ion channels, the regulation of respiration in response to acid-base disturbances, and the response to ischemia and anoxia and other insults.